Coherent Raman microscopy has emerged as an important label-free imaging modality, which can enable long duration non-invasive imaging of cells and tissues. However, current coherent Raman systems require scanning to obtain three-dimensional images, hindering studies of rapid dynamic biological processes. Additionally, coherent Raman imaging systems usually rely on complex and expensive laser sources, which limit their accessibility. In this Phase I SBIR study, Atoptix, LLC. proposes to develop a fiber-based holographic coherent anti-Stokes Raman scattering (CARS) microscope to overcome these limitations of scanning and system complexity. The key innovation, CARS holography, merges the unique capabilities of CARS spectroscopy and holographic imaging by recording of a digital CARS hologram which captures both the amplitude and the phase of an anti-Stokes image signal. CARS holography can thus perform label-free, 3D imaging without scanning, and have an improved sensitivity over conventional CARS due to its homodyne detection nature. Furthermore, the proposed system will use a robust, turn-key fiber laser based excitation system which overcomes the complexities and high costs associated with existing coherent Raman systems. To perform this work, Atoptix will collaborate with Penn State University researchers who are the inventors of CARS holography. Specific Aim 1 is to develop a synchronized two-color ultrafast fiber laser system that will provide the CARS pump/probe and Stokes excitation sources. Specific Aim 2 is to develop the prototype holographic CARS microscope. The system will be characterized and optimized to assess performance and capability for Phase II development of an advanced prototype system.